Real time DNA amplification and detection methods are efficient for sequence identification and quantification of a target since no pre-hybridization amplification is required. Amplification and hybridization are combined in a single step and can be performed in a fully automated, large-scale, closed-tube format.
Methods that use hybridization-triggered fluorescent probes for real time PCR are based either on a quench-release fluorescence of a probe digested by DNA Polymerase (e.g., methods using TaqMan, MGB-TaqMan) or on a hybridization-triggered fluorescence of intact probes (e.g., molecular beacons, and linear probes, see U.S. Pat. Nos. 6,030,787, U.S. Pat. No. 5,723,591). In general, the probes are designed to hybridize to an internal region of a PCR product during annealing stage. For those methods utilizing TaqMan and MGB-TaqMan the 5′-exonuclease activity of the approaching DNA Polymerase cleaves a probe between fluorophore and quencher thus releasing fluorescence.
What is needed in the art are new oligonucleotide probes and conjugates that are useful in “real-time” amplification processes and which can be prepared with a variety of nucleotide bases in short lengths. The present invention provides such probes and conjugates, as well as methods for their use.